Alkaline phosphatase for treating an inflammatory disease of the gastro-intestinal tract

ABSTRACT

The present invention provides a use for alkaline phosphatase for the manufacture of a medicament for the prevention or reduction of toxic LPS influx through a mucosal lining of a mammalian body cavity. A source of alkaline phosphatase is administered for the prophylaxis or treatment of LPS mediated or exacerbated diseases. The invention also provides compositions comprising a source of alkaline phosphatase for the prevention or reduction of (toxic) LPS influx or passage through mucosal layers.

This Application is a National Stage Application filed under Rule 371based on PCT/NL2005/000084 filed Feb. 4, 2005 which claims benefit toProvisional Application 60/541,363 filed Feb. 4, 2004.

FIELD OF THE INVENTION

The current invention relates to the field of medicine and in particularto the use of LPS detoxifying and neutralizing enzymes. The presentinvention also relates to the field of pharmacy and in particular to thepharmaceutical use of alkaline phosphatase enzymes.

BACKGROUND OF THE INVENTION

Lipopolysaccharides (LPS; also referred to as endotoxins) are present inthe cell walls of Gram-negative bacteria. When LPS is presented to avertebrate body it stimulates the innate and cellular immune responsesin a wide variety of cell types. The production of cytokines andchemokines (such as TNF's, various interleukines, interferons andothers) will attract and activate cells of the immune system, which mayculminate ultimately in an LPS induced systemic inflammatory responsesyndrome (SIRS) under certain conditions.

LPS or endotoxins are toxic to most mammals and regardless of thebacterial source, all endotoxins produce the same range of biologicaleffects in the animal host. The injection of living or killedGram-negative cells, or purified LPS, into experimental animals causes awide spectrum of non-specific pathophysiological reactions such as:fever, tachycardia, tachypneu, hyper or hypothermia, changes in whiteblood cell counts, disseminated intravascular coagulation, hypotension,organ dysfunction and may even result in shock and death.

Injection of small doses of endotoxin results in a proinflammatoryresponse in most mammals, but the dose response range and steepnessthereof varies significantly with the species and even within speciesmay differ significantly depending on e.g. LPS-tolerance. The sequenceof pro-inflammatory events follows a regular pattern (inflammatorycascade): (1) latent period; (2) physiological distress (diarrhea,prostration, shock); and in case of severe septic shock and multipleorgan failure may result in (3) death. How soon death occurs varies onthe dose of the endotoxin, route of administration, and species ofanimal.

The physiological effects of endotoxin are mainly mediated by the lipidA-moiety of LPS. Since Lipid A is embedded in the outer membrane ofbacterial cells, it only exerts its toxic effects when released frommultiplying cells in a soluble form, or when the bacteria are lysed as aresult of autolysis, complement and the membrane attack complex (MAC),ingestion and killing by phagocytes, or killing with certain types ofantibiotics. LPS released into the bloodstream can be neutralised bymany blood components to a certain degree, amongst which several plasmalipids and proteins, among which LPS-binding proteins. The LPS-bindingprotein complex interacts with CD14 and Toll like receptors on monocytesand macrophages and through other receptors on endothelial cells. Inmonocytes and macrophages three types of events are triggered duringtheir interaction with LPS:

Firstly, production of cytokines, including IL-1, IL-6, IL-8, tumornecrosis factor (TNF) and platelet-activating factor. These in turnstimulate production of prostaglandins and leukotrienes. These arepowerful mediators of inflammation and septic shock that accompaniesendotoxin toxemia. LPS activates macrophages to enhanced phagocytosisand cytotoxicity. Macrophages are stimulated to produce and releaselysosomal enzymes, IL-1 (“endogenous pyrogen”), and tumor necrosisfactor (TNFalpha), as well as other cytokines and mediators.

Secondly, activation of the complement cascade. C3a and C5a causehistamine release (leading to vasodilation) and effect neutrophilchemotaxis and accumulation. The result is inflammation.

Finally, activation of the coagulation cascade. Initial activation ofHageman factor (blood-clotting Factor XII) can activate several humoralsystems resulting in coagulation: a blood clotting cascade that leads tocoagulation, thrombosis, acute disseminated intravascular coagulation,which depletes platelets and various clotting factors resulting ininternal bleeding and also activation of the complement alternativepathway (as above, which leads to inflammation). Plasmin is activatedwhich leads to fibrinolysis and hemorrhaging and kinin activationreleases bradykinins and other vasoactive peptides which causeshypotension. The net effect is induction of inflammation, intravascularcoagulation, hemorrhage and shock.

LPS also acts as a B cell mitogen stimulating the polyclonaldifferentiation and multiplication of B-cells and the secretion ofimmunoglobulins, especially IgG and IgM.

The physiological activities of LPS are mediated mainly by the Lipid Acomponent of LPS. Lipid A is a powerful biological response modifierthat can stimulate the mammalian immune system. During infectiousdisease caused by Gram-negative bacteria, endotoxins released from, orpart of, multiplying cells have similar effects on animals andsignificantly contribute to the symptoms and pathology of the diseaseencountered. The primary structure of Lipid A has been elucidated andLipid A has been chemically synthesized. Its biological activity appearsto depend on a peculiar conformation that is determined by theglucosamine disaccharide, the PO₄ groups, the acyl chains, and also theKDO-containing inner core of the LPS molecule.

Alkaline phosphatase (AP), has been described earlier as a key enzyme inthe dephosphorylation of LPS (endotoxin) under physiological conditionsboth in vitro and in vivo as a natural response to detoxify andneutralise LPS (U.S. Pat. No. 6,290,952, Poelstra et al., Am J Pathol.1997 October; 151(4):1163-9).

Reports on the enzyme activity of AP involve its extremely high pHoptimum for the usual exogenous substrates and its localization as anecto-enzyme. Endotoxins are molecules that contain several phosphategroups and are usually present in the extracellular space. AP is able todephosphorylate this bacterial product at physiological pH levels, byremoving phosphate groups from amongst others the toxic lipid A moietyof LPS. As phosphate residues in the lipid A moiety determine thetoxicity of the molecule, the effect of the AP inhibitor levamisole invivo using a septicemia model in the rat confirmed the specificity of APfor LPS containing phosphate groups (Poelstra et al., 1997). The resultsdemonstrated that inhibition of endogenous AP by levamisolesignificantly reduces survival of rats intraperitoneally injected withE. coli bacteria, whereas this drug does not influence survival of ratsreceiving a sublethal dose of the gram-positive bacteria Staphylococcusaureus, illustrating a crucial role for this enzyme in host defense. Theeffects of levamisole during gram-negative bacterial infections and thelocalization of AP as an ecto-enzyme in most organs as well as theinduction of enzyme activity during inflammatory reactions andcholestasis is in accordance with such a protective role.

The prime source of LPS exposure in the human body are the gram negativemicroorganisms that live within the human digestive or gastrointestinal(GI) tract. There are far more bacteria in the digestive system thanthere are on the skin or other parts of the body, making the GI tractand GI mucosa the main route of entry for LPS into the circulation. Anaverage adult carries about 100 trillion bacteria in the intestines,most of which locate in the colon, contributing to 1-1.5 kg of his bodyweight. There are more than 400 species of bacteria found in thedigestive system. These include both beneficial (commensal) andpotentially harmful (pathogenic) species, which continually compete tomaintain a well-balanced intestinal flora.

Mucosal surfaces, and in particular (but not limited to) the intestinalmucosa, are exposed to this wide variety of commensal and potentiallypathogenic bacteria, among which many gram negative endotoxin/LPSproducing, Gram-negative bacteria such as E. coli, Salmonella, Shigella,Pseudomonas, Neisseria, Haemophilus, Helicobacter, Chlamydia and otherleading pathogens. The intestinal epithelium is of particular importanceas it forms a dynamic barrier that regulates absorption of nutrients andwater and at the same time restricts uptake of microbes and othernoxious materials such as LPS from the gut lumen.

It is well established that a major fraction of LPS influx from thelumen of the gut through the mucosal lining into the circulation of avertebrate body is mediated through chylomicrons (Harris et al., 1998,2000, 2002). Coincidental with ingestion of lipids and chylomicronintroduction in circulation, capable of carrying LPS, a significantincrease in lymphatic AP derived from the GI-tract is reported (Nauli etal., 2002). LPS-influx through the GI-barrier is increased normally witha saturated fat-rich diet. LPS inserts with its lipid A acyl chain intolipoprotein phospholipids. Thereby LPS passes the intestinal barrier byco-migrating with chylomicrons, that are taken up predominantly at thesmall intestines ileum (Harris et al., 2002). After a fat rich foodintake a significant rise of glycosyl-phosphatidyl-inositol(GPI)-anchored AP complexed to lipoproteins is detected in lymph as well(Nauli et al., 2003).

The physiological roles of—and the interpretation of AP serum levels arenot clear, but a role in detoxification of LPS has emerged from currentresearch. The co-presence of both AP and LPS in chylomicron richfractions suggest a role for AP in dephosphorylating the gut derived-LPSalready at close vicinity. Detoxification can take place both in theintestinal lumen or en-route to or upon presentation to the liver,specifically in this context to Kuppfer cells and the hepatocytes, whichclear the chylomicrons from circulation.

Increased serum AP levels are associated with hepatic damage. Upon anendotoxin insult, circulatory AP is redirected to hepatocytes, therebyreducing circulating AP levels initially (Bentala et al., 2002) throughreceptor-mediated uptake (asialo-glycoprotein receptor). Hepatocytesalso remove the LPS-loaded chylomicrons (Harris et al., 2002) rapidlyfrom circulation with a half life of 5-10 minutes. LPS is next removedthrough biliary excretion, thereby preventing Kupffer cells, being amajor target for circulating LPS to become activated (Harris, 2002).Bentala et al., 2002, showed that Kupffer cells accumulate AP inLPS-insulted animal models as well. This may imply that under normalconditions Kuppfer cells will not be activated since LPS (lipidAmoiety), or its derivative MPLS (MPLA, derivative from Lipid A), isprimarily presented to hepatocytes through a lipoprotein receptor andnext is removed via biliary secretion. However under conditions withexcess LPS, Kupffer cells are activated through a TLR-4 (LPS) receptor.

A wide array of animals have AP and several other entities present tocounteract a (bacterial) insult, either local or systemic, induced oravailable as guard/watchdog function. Amongst others activatedneutrophils or macrophages express a wide array of inflammatorymediators destined to neutralise the insult. Moieties like, but notrestricted to LPS binding protein (LBP), CD14, Apo-E, VLDL, HDL,albumin, immunoglobulin and AP all have been described to serve thisfunction. When such an insult however is not overcome, e.g. in case of asevere Gram negative or positive insult, resulting inflammatorymediators may initiate a systemic inflammatory response syndrome (SIRS).

It was postulated that AP is consumed as a consequence of its catalyticaction towards LPS (Poelstra et al., 1997). This implies thatsubsequently normal levels are to be restored through a controlledmechanism. In patients suffering from septicaemia, it has been observedthat increased serum AP may be preceded by reduced AP serum levels(Manintveld and Poelstra, patent application EP 989626940) and thatcirculating AP would be cleared from circulation upon LPS interaction(Bentala et al., 2002). The increase in subsequent AP-levels thereforemay be a feedback mechanism in response to this AP reduction. Amechanism for such a LPS/AP responsiveness has not been depictedto-date.

In inflammatory processes (temporary) increases are found for serum AP.In the context of this invention such an increase of AP is regarded as anatural response of the innate immune system to an LPS insult to tacklethese insults and restore natural balance. Increased AP plasma levelsare the result of massive shedding of AP from hepatocytes in response tothe LPS insult. It has been observed that LPS induces Phospholipase-Dactivity (Locati et al., 2001) which in turn has been reported to actupon GPI anchored proteins, amongst which AP (Deng et al., 1996) ande.g. CD14, thereby effectively shedding the proteins into circulation(Zhang F et al., 2001, Locati M. et al., 2001).

Circulating plasma AP—predominantly anchorless livertype AP (Ahn et al.,2001)—may thus already have exerted its LPS detoxificating activity atthe plasma membrane surface and is subsequently freed from thehepatocyte membrane into circulation prior to its subsequent eliminationfrom circulation by e.g. the asialo glycoprotein-route.

AP exerts its catalytic activity towards LPS primarily in the vincinityof a membrane, possibly in so-called lipid rafts (drm ordetergent-resistant membrane fraction) where it has been reported toreside. Several publications favor such a catalytic activity of AP at amembrane surface, either presented at the tissue level or released intocirculation like with circulating liver plasma membrane fragments (LPMF)(e.g. Deng et al., 1996). The increased AP levels observed inchronically inflamed patients may be caused by the suboptimaldetoxification of the gut-derived influx of LPS, which is often enhancedunder pathological conditions prior to mobilization of hepatic AP.

The treatment of inflammatory diseases accounts for a substantialpercentage of the gross medical cost in developed countries and theincidence of these inflammatory diseases is continuously rising due tokey factors like ageing of the population and an increasing number ofpatients having suppressed immune systems as a consequence of medicationand treatment of a wide array of diseases like heart disease,auto-immunity disorders and allergies, organ transplantations, cancerchemo- or radiotherapy and infectious diseases like AIDS. To a certainextent these diseases relate to an influx of bacterial LPS. The influxof LPS is often enhanced by a medical condition of a subject, causing aninflammatory process by a malfunctioning or non-balanced innate immunesystem, which constitutes the first line of defense against e.g.microbial insults, in particular from LPS/endotoxin producing bacteria.

The current invention is aimed at providing new methods and compositionsfor the detoxification, neutralisation or complexation of LPS in situ atmucosal tissues in body cavities before LPS can pass through the mucosallayer and enter the circulation where it would elicit toxic effectsand/or an inflammatory response.

DESCRIPTION

Definitions

Endotoxins are part of the outer membrane of the cell wall ofGram-negative bacteria. Endotoxins are invariably associated withGram-negative bacteria whether the organisms are pathogens or not.Although the term “endotoxin” is occasionally used to refer to anycell-associated bacterial toxin, it is properly reserved to refer to thelipopolysaccharide or LPS complex associated with the outer membrane ofGram-negative bacteria such as Escherichia (E. coli), Salmonella,Shigella, Pseudomonas (Ps. aeruginosa), Neisseria (N. meningitidis),Haemophilus (H. influenzae), Chlamydia (Chl. pneumoniae), Helicobacter(H. pylori) and other leading pathogens.

Lipopolysaccharides are complex amphiphilic molecules with a monomericmolecular weight of about 10 kDa, that vary widely in chemicalcomposition both between and among bacterial species. LPS consists ofthree components or regions, Lipid A, an R polysaccharide and an Opolysaccharide. Lipid A contains the hydrophobic, membrane-anchoringregion of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine(NAG) dimer with 6 or 7 fatty acids (FA) attached. The Core (R) antigenor R polysaccharide is attached to the 6 position of one NAG. The Rantigen consists of a short chain of sugars. Two unusual sugars areusually present, heptose and 2-keto-3-deoxyoctonoic acid (KDO), in thecore polysaccharide. KDO is unique and invariably present in LPS and sohas been an indicator in assays for LPS (endotoxin).

With minor variations, the core polysaccharide and lipid A is common toall members of a bacterial genus (e.g. Salmonella), but it isstructurally distinct in other genera of Gram-negative bacteria.Salmonella, Shigella and Escherichia have similar but not identicalcores.

The biological activity of endotoxin is associated with thelipopolysaccharide (LPS). Toxicity is associated with the lipidcomponent (Lipid A) and immunogenicity is associated with thepolysaccharide components. The cell wall antigens (O antigens) ofGram-negative bacteria are components of LPS. LPS elicits a variety ofinflammatory responses in an animal. Because it activates complement bythe alternative (properdin) pathway, it is often part of the pathologyof Gram-negative bacterial infections.

The Limulus assay (LAL) is a well known bioassay in the art to measureLPS concentrations and toxicity. The assay is based on an exquisitelysensitive primitive defense system of the ancient horseshoe crab,Limulus polyphemus. An assay based on this system can be measured by acolor change after cleavage of chromogenic or fluorogenic substrates.LAL can used to measure sub-picogram quantities of these microbialproducts very rapidly with minimal equipment and can detect live, deadand non-cultivable organisms. The blood cells of Limulus, or amebocytes,of the horseshoe crab constitute a primitive “innate” immune defense,binding to the outer cell wall structures of the microbial cell andcausing a blood clotting reaction. Soluble LPS, as well as cell wallcomponents of other microbes, such as beta glucans in yeast and fungi,have been shown to cause the horseshoe crab blood to clot. This clottingreaction is now known to be an enzyme cascade whose components arepresent in granules within the amebocyte. A lysate of the amebocyte isproduced by collecting blood cells in a sterile, endotoxin-free methodand is available as a commercial product (LAL, Charles River Endosafe,Charleston, S.C.) currently used as an assay for LPS and detoxificationof LPS by AP enzymes and compositions comprising sources of AP.

Alkaline phosphatase (AP): EC 3.1.3.1 according to IUBMB EnzymeNomenclature, the common name is alkaline phosphatase (AP), an enzymethat catalyzes the reaction of a phosphate monoester+H₂O=analcohol+phosphate. Other name(s) for AP are alkalinephosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkalinephosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoesterphosphohydrolase (alkaline optimum). The systematic name of AP isphosphate-monoester phosphohydrolase (alkaline optimum).

AP is a wide specificity enzyme, it also catalysestransphosphorylations. In humans and other mammals, at least fourdistinct but related alkaline phosphatases are known. They areintestinal, placental, placental-like, and liver/bone/kidney (or tissuenon-specific) alkaline phosphatase. The first three are located togetheron chromosome 2 while the tissue non-specific form is located onchromosome 1. The exact physiological functions of the APs are notknown, but AP appears to be involved with a large number ofphysiological processes, among which the detoxification of LPS throughdephosphorylation of the toxicity determining lipid A moiety of LPS. Forthe current invention, the term alkaline phosphatase may comprise anyenzyme exhibiting detoxification of LPS as determined by a Limulus assayor another bioassay. The activity of an AP enzyme or composition orpreparation comprising AP can be determined by detoxification ofcommercially available LPS (for instance Lipopolysaccharide (LPS) fromSigma, Cat. No. L-8274) in vitro, followed by a standard Limulus assay(LAL) before and after AP treatment. Alternatively LPS toxicityreduction through AP activity can be quantitated by means of a bioassayas described by Beumer et al., 2003.

Mucosa is a mucus-secreting membrane lining all body cavities orpassages that communicate with the exterior. Mucosa is a moist tissuethat lines many organs (such as the intestines) and body cavities (suchas nose, mouth, lungs, vagina, bile duct, esophagus) and secretes mucous(a thick fluid). The mucosa, or mucous membrane, is a type of tissueprotects body cavities from environmental conditions, pathogens andtoxic substances and are usually moist tissues that are bathed bysecretions (such as secretions in the bowel, lung, nose, mouth andvagina).

DETAILED DESCRIPTION OF THE INVENTION

The current invention is aimed at providing new methods and compositionsfor the detoxification of LPS in situ at mucosal tissues in bodycavities. A first aim of the in situ detocification of LPS at mucosalsurfaces in the body is to prevent or reduce local inflammatory responseat such surfaces. Furthermore, the LPS that is thus detoxified is nolonger available for passage through mucosal layers and thus cannotenter the circulation where it will exert its toxic effects and/or causea further local and/or systemic inflammatory response. Detoxificationmay also comprise neutralising or complexation of LPS by AP, which byclose proximity may form a detoxified composition. The methods comprisethe use of sources of alkaline phosphatase, which is known to be apotent means for LPS detoxification. A source of AP can be any APenzyme, or any composition comprising the AP enzyme and any means whichis capable of producing a functional AP enzyme in the context of thecurrent invention, such as DNA or RNA nucleic acids encoding an APenzyme. The nucleic acid encoding AP may be embedded in suitable vectorssuch as plasmids, phagemids, phages, (retro)viruses, transposons, genetherapy vectors and other vectors capable of inducing or conferringproduction of AP. Also native or recombinant micro-organisms, such asbacteria, fungi, protozoa and yeast may be applied as a source of AP inthe context of the current invention.

In a first embodiment the invention provides a method for the preventionor reduction of toxicity LPS at a mucosal lining of a mammalian bodycavity comprising the step of administering a source of AP at themucosal layer. For those jurisdictions where methods of treatment areunpatentable by law, the invention likewise pertains to the use of AP asdefined above, or the use of a composition containing a source ofalkaline phosphatase as defined above. The source of AP is used for themanufacture of a medicament for delivery of AP at a mucosal layer forthe prevention or reduction of toxic LPS influx through a mucosal liningof a mammalian body cavity. In an additional embodiment the inventionprovides a method for the prevention or reduction of toxic LPS influxthrough a mucosal lining of a mammalian body cavity comprising the stepof administering a source of AP at the mucosal layer.

In particular the above mentioned method of administering a source of APat mucosal layers of body cavities is suited for the treatment orprofylaxis of LPS mediated or exacerbated diseases, although the methodmay also be advantageously used for healthy subjects as a prophylactictreatment aimed at the prevention of LPS induced toxicity and/or LPSinduced or exacerbated diseases. The beneficial effects of APadministration to reduce toxic LPS levels in body cavities and atmucosal layers according to the current invention will generate ageneral health promoting effect regardless of the medical condition ofthe subject treated. The health promoting effect may be furtheraugmented by the consequent decrease in LPS influx through mucosallayers. An LPS mediated or induced disease may be any disease, symptomor group of symptoms caused by LPS toxicity. An LPS exacerbated diseasemay be any disease or symptom that is not directly caused by LPS or LPStoxicity but a disease which symptoms and clinical features may beaggravated by LPS and the clinical state of the subject suffering fromsuch a disease is worsened by LPS and LPS toxicity.

Preferably the method is aimed at the treatment of an LPS mediated orexacerbated diseases selected from the group consisting of: inflammatorybowel diseases, sepsis/septic shock, systemic inflammatory responsesyndrome (SIRS), Meningococcemia, trauma/hemorrhagic shock, burninjuries, cardiovascular surgery/cardiopulmonary bypass, liversurgery/transplant, liver disease, pancreatitis, (necrotising)enterocolitis, periodontal disease, pneumonia, cystic fibrosis, asthma,coronary heart disease, congestive heart failure, renal disease,hemolytic uremic syndrome, kidney dialysis, autoimmune diseases, cancer,Alzheimer, rheumatoid arthritis, lupus, systemic lupus erythematosus.

Circulating endotoxin has been detected in patients with inflammatorybowel diseases, in particular in patients diagnosed with Crohn's diseaseand ulcerative colitis. Its presence is the consequence of the damagedintestinal mucosa and increased LPS influx or gut translocation andcauses or exacerbates the inflammatory response in the intestines.Intestinal bacterial translocation and LPS gut translocation is alsoobserved in acute pancreatitis and liver diseases caused by cirrhosis,alcohol abuse, obstructive jaundice and other hepatic conditions.Endotoxin has also been implicated in the development of periodontaldisease, where it penetrates the gingival epithelium/mucosa, ensuing alocal inflammatory response. In a preferred embodiment the methodcomprises oral administration of a source of AP to reduce LPS toxicityat and/or passage of LPS through the mucosa.

The preferred mode of administration comprises the use of pharmaceuticalcompositions comprising sources of AP, which may be delivered in a dailydoses regimen to reduce toxic LPS levels in the lumen of the GI tractfor a prolonged period of time. Preferably the pharmaceuticalcompositions comprise an enteric coating to protect AP from thedetrimental effects of gastric juices (pH 1.0 to 2.5) and ensureefficient delivery of AP at the mucosa of the intestinal tract. Morepreferably, the pharmaceutical composition is a source of AP comprisedwithin an enteric coat.

Enteric coatings arrest the release of the active compound from orallyingestible dosage forms. Depending upon the composition and/orthickness, the enteric coatings are resistant to stomach acid forrequired periods of time before they begin to disintegrate and permitslow release of AP (drug) in the lower stomach or upper part of thesmall intestines. Examples of some enteric coatings are disclosed inU.S. Pat. No. 5,225,202 (incorporated by reference). Examples of entericcoatings comprise beeswax and glyceryl monostearate; beeswax, shellacand cellulose, optionally with neutral copolymer of polymethacrylicacidesters; copolymers of methacrylic acid and methacrylic acid methylestersor neutral copolymer of polymethacrylic acid esters containing metallicstearates (for references enteric coatings see: U.S. Pat. Nos.4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560,4,524,060, 5,536,507). Most enteric coating polymers begin to becomesoluble at pH 5.5 and above, with a maximum solubility rates at pH above6.5. Enteric coatings may also comprise subcoating and outer coatingsteps, for instance for pharmaceutical compositions intended forspecific delivery in the lower GI tract, i.e. in the colon (pH 6.4 to7.0, ileum pH 6.6), as opposed to a pH in the upper intestines, in theduodenum of the small intestines the pH ranges 7.7-8 (after pancreaticjuices and bile addition). The pH differences in the intestines may beexploited to target the enteric-coated AP composition to a specific areain the gut. It also allows the selection of a specific AP enzyme that ismost active at a particular pH in the intestine. For instance CIAP (calfintestinal AP) and human placenta (HPLAP) AP are most active at alkalinepH 8.2 in the smallintestinal duodenum, jejunum and ileum, whereas milkderived AP and Bone/Liver/Kidney or Tissue non specific AP (TSN AP) aremost active at neutral pH and better suited for treatment of the colon(pH 7.4).

The most preferred mucosal tissues to be treated according to thecurrent invention are the mucosal tissues lining the intestinal tractbody cavities. Orally administered AP is delivered at the mucosaltissues of the GI tract, which comprises the esophagus, stomach, thesmall intestines or bowel, (duodenum, jejunum, ileum) and largeintestines or colon (caecum, ascending colon, transverse colon,descending colon, sigmoid colon, rectum and anus). Within the scope ofthe current invention, also mucosal tissues lining the mouth, the ductsof the bile and the pancreas are part of the intestinal tract and may betreated according to method of the current invention.

The compositions comprising a source of AP according to the currentinvention are particularly suited for oral administration to preventtreat, reduce, treat or alleviate inflammatory diseases of thegastrointestinal tract. Inflammatory diseases of the gastrointestinaltract may be induced and/or exacerbated significantly by the influx ofLPS. A reduction in the amount of toxic LPS in the lumen of theintestines by administration of sources of AP will, throughdetoxification of the lipid A moiety of LPS, result in a correspondingdecrease in the systemic influx of toxic LPS in the circulation of asubject. In a most preferred embodiment, the oral administration ofsources of AP are particularly preferred for the prophylaxis ortreatment of the following inflammatory disease of the gastrointestinaltract: Crohn's disease, colitis, (necrotizing) enterocolitis, colitisulcerosa, hepatobiliary disease, hepatitis B, hepatitis C, livercirrhosis, liver fibrosis, bile duct inflammation, biliary obstruction,pancreatitis, acute pancreatitis, peritonitis and periodontal disease.

In another embodiment of the invention, a source of AP is orallyadministered to subjects who suffer from an increased mucosalpermeability of the gastrointestinal tract. Increased mucosalpermeability of the GI tract is often the result of a decreasedperfusion or ischemia of the intestines. Ischemia, a lack of oxygensupply by the bloodstream, may be caused by heart failure, injuries,trauma or surgery. Ischemia of the intestines results in amalfunctioning of the mucosa and a consequential increase in the influxor translocation of toxic LPS from the gut, resulting in both local andsystemic toxicity and inflammation. The toxicity and inflammatoryresponse may even further enhance the mucosal permeability, resulting ina vicious circle. Increased mucosal permeability of the GI tract may bethe result of inflammatory bowel diseases or other pathologicalconditions of the GI tract. Oral administration of sources of APaccording to the current invention will significantly reduce or abolishthis increased influx of toxic LPS by detoxification of LPS in the lumenof the intestinal cavities. Exogenous administration of AP will breakthe vicious circle of LPS influx through the mucosa, inflammation andenhanced permeability of the mucosa resulting in an enhanced LPS influx.Decreased perfusion or ischemia of the intestines and a concomitantincreased LPS influx is observed by the following group of diseases orconditions: burns, trauma and/or wounds which may result from accidents,gunshot or knife wounds, surgery, and in particular surgery withcardiopulmonary bypass. Also malfunctioning of the heart function, suchas congenital heart disease, congestive heart failure, coronary heartdisease and ischemic heart disease may result in ischemia of theintestines and an increased influx of LPS. It is a preferred embodimentof the current invention to treat subjects suffering from this group ofdiseases and conditions with timely and regular oral administration ofcompositions comprising a source of AP to prevent or reduce LPS influxthrough the intestinal mucosa with an enhanced permeability for LPS.

In another embodiment the current invention is aimed at providing asource of AP at the mucosal lining the respiratory tract. Therespiratory tract is another body cavity with a mucosal lining that isexposed to the toxic effects of LPS. LPS, either free or associated withinhaled bacteria, enters the respiratory tract bronchial and pulmonarymucosa via normal respiration, by inhalation of e.g. dust-particles, orfrom infections of the respiratory tract and mucosal tissues with gramnegative bacteria. In addition, tobacco is known to be a rich source ofLPS and smoking, either passive or active, may further contributesignificantly to the LPS burden of the bronchial and pulmonary mucosa.Under normal conditions this LPS is detoxified by the local mucosalimmune defence system in the respiratory tract. Therefore, in anotherpreferred embodiment the current invention pertains to theadministration of a source of AP via inhalation to the bronchial andpulmonary mucosa to prevent or reduce LPS influx through the mucosa ofthe respiratory tract for those conditions where the normal defenseresponses to LPS are malfunctioning. The current invention also providescompositions suitable for the delivery of AP at the bronchial andpulmonary mucosa. These compositions are preferably administered to forthe prophylaxis or treatment of inflammatory diseases of the respiratorytract. In a most preferred embodiment pulmonary administration of asource of AP according to the current invention is applied to treat orprevent a disease selected from the group consisting of pneumonia, lunginfections, asthma, CARA, cystic fibrosis, bronchitis and emphysema. Thecurrent invention also provides spraying devices, loaded with acomposition comprising a source of AP and optionally various excipientssuch as propellants, carriers, nebulisers and/or diffusers, suitable forthe administration of AP at the pulmonary and bronchial mucosa. Sprayingdevices, inhalators and nebulisers are known in the art ofpharmaceutical formulation and will be obvious to the skilled artisan,reference Remmington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia Pa., 17th ed. 1985.

In yet another embodiment, the current invention is aimed at the topicaladministration of a source of AP at a mucosal layer lining a bodycavity. In a preferred embodiment the body cavity is the nasal cavity,oral cavity, vagina or rectum. Topical administration of a source of APat a mucosal tissue lining a body cavity is preferably applied to treatlocal or systemic inflammatory diseases, and it is particularlypreferred for the treatment or prophylaxis of infections of the nasal,vaginal, oral or rectal cavities, sexually transmitted diseases andinfections, urinary tract infections, bladder infections and periodontaldisease.

The current invention also provides compositions comprising a source ofAP, amongst which are pharmaceutical and nutraceutical compositionscomprising a source of AP. The compositions may optionally comprisepharmaceutically acceptable excipients, stabilizers, activators,carriers, permeators, propellants, desinfectants, diluents andpreservatives. Suitable excipients are commonly known in the art ofpharmaceutical formulation and may be readily found and applied by theskilled artisan, references for instance Remmington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia Pa., 17th ed. 1985. In apreferred embodiment the compositions comprising a source of AP aresuitable for oral administration and comprise an enteric coating toprotect the AP from the adverse effects of gastric juices and low pH.Enteric coating and controlled release formulations are well known inthe art (references as described above). Enteric coating compositions inthe art may comprise of a solution of a water-soluble enteric coatingpolymer mixed with the active ingredient(s) such as AP and otherexcipients, which are dispersed in an aqueous solution and which maysubsequently be dried and/or pelleted. The enteric coating formed offersresistance to attack of AP by atmospheric moisture and oxygen duringstorage and by gastric fluids and low pH after ingestion, while beingreadily broken down under the alkaline conditions which exist in thelower intestinal tract.

AP containing compositions for the delivery of AP at mucosal tissues fordetoxification of LPS according to the current invention preferablycomprise an eukaryotic AP, more preferably a mammalian AP, which may beof the types tissue non-specific AP, such as liver-bone or kidney type,or tissue specific such as placental AP and intestinal AP. Mostpreferably the mammalian AP is a human or a bovine AP.

In a preferred embodiment of the current invention the source of AP isAP which is preferably produced or isolated from milk, preferably bovinemilk. The milk may be obtained from animals that have been bred orgenetically modified to produce elevated levels of AP in their milk ascompared to wild-type animals. The preparation of AP enriched fractionsfrom milk is known in the art. For instance the milkfat globule membraneenriched or derived fraction is the preferred AP enriched milk fractionand may be routinely obtained by conventional skimming of raw milk. APisolated from milk may be formulated in pharmaceutical compositions andin food compositions or in nutraceuticals.

In a preferred embodiment the AP containing composition for oraladministration of AP to the mucosa of the gastrointestinal tractaccording to the current invention is a food product or nutraceuticalenriched for AP. In one embodiment the food product may be a plant,fruit or vegetable, optionally genetically modified to contain anenhanced level of AP. In another embodiment the AP containing foodproduct or nutraceutical is a dairy product. In particular preparationsand compositions containing non-pasteurised milk or fractions thereof,preferably bovine milk, contain high levels of AP and are particularlysuited for oral administration as a source of AP according to thecurrent invention.

The current invention also pertains to a method for the preparation ofan AP enriched dairy product, preferably milk, a milk fraction or milkproduct. The method comprises the fractionation of raw milk, preferablybovine milk, pasteurisation of the fractions not containing or not richin AP and reformulating said fractions with the unpasteurised, AP richfractions, to obtain a less perishable and AP enriched dairy product.The non pasteurised AP rich fractions may be sterilised by other means,such as, but not limited to, irradiation with UV-, X- or gamma-rays,filtration, pressure, osmotic pressure, chemicals or antibiotics,ensuring that the AP enzyme remains substantially active and that themilkfraction becomes substantially sterile. This dairy product may beused in compositions or administered directly to subjects suffering fromor at risk of developing an LPS mediated or exacerbated disease and/orinflammation. However, the AP enriched dairy product may also be offeredto healthy subjects as a pharmaceutical or nutraceutical product for thereduction of toxic LPS in the gastrointestinal tract and for thereduction of LPS influx through the gastrointestinal mucosa.

EXAMPLES Example 1

The current invention, and in particular the effectiveness of APenzymes, preparations and compositions, and different modes ofadministration of AP may be tested in various animal models forinflammatory bowel diseases that are known in the art. Animal modelsmimicking human IBD comprise antigen-induced colitis and colitis inducedby microbials; other inducible forms of colitis, chemical (for instancetrinitrobenzene sulphonic acid (TNBS) in Montfrans et al., 2002),immunological and physical and genetic colitis models (transgenic andknock-out models, see for instance SCID-mice, Davis et al., 2003, IL-10KO mice, Rennick et al., 2000, SAMP1/Yit mouse, Kosiewicz, et al., 2001and Strober et al., 2001); adoptive transfer models and spontaneouscolitis models (Kosiewicz, M. M. et al., 2001).

The chemically induced Dextran Sulphate Sodium (DSS) colitis model wasoriginally described by Okayasu et al; Gastroenterology, 1990: 98,694-702, and is a model for human ulcerative colitis. The modelcomprises acute and chronic ulcerative colitis in mice caused byadministration of 3-10% DSS in their drinking water. The morphologicalchanges and changes in the intestinal microflora are similar to thoseseen in clinical cases of ulcerative colitis. The colon damage developsdue to a toxic effect of DSS on the epithelial cells and to phagocytosisby lamina propria cells, resulting in production of TNF-alpha andIFN-gamma.

Experimental Design for Acute DSS Colitis:

DSS (MW 40.000 obtained from ICN chemicals) is dissolved in acidifieddrinking water in a concentration of 5% (w/v) and given ad libitum tofemale balb/c mice (Harlan). The solution is refreshed every day. After7 days of treatment, treated and control mice may be sacrificed and theintestines analysed. The total colon is dissected (from caecum torectum) and its length is recorded. About half of the colon is frozen inliquid nitrogen and cryo sections are made for morphology (HE staining).Also small parts of the spleen and the liver are snap-frozen in liquidnitrogen for immunohistochemical purposes. A small part of the colon isused to prepare tissue homogenates for cytokine measurements. Smallcolon strips are cultured in RPMI/10% FCS for 24 h in absence orpresence of LPS. Cytokine secretion (TNF alpha; IL-1 beta; IFN gamma) inthe supernatant is measured using specific ELISA assays. Spleen andmesenteric lymph-nodes are dissected and squeezed to prepare single cellsuspensions. 4 Peyer's Patches near the colon are dissected and singlecell suspensions are made by use of collagenase. Cells are characterizedusing flow-cytometric techniques. Spleen cells are cultured for 24 h inRPMI/10% FCS in absence or presence of LPS or Con A. Cytokine secretion(TNF alpha; IL1 beta; IFN gamma) in the supernatant is measured usingspecific ELISA assays. Feaces are collected and cultured on McConkeyagar plates for enterobacteriaceae contents. For total aerobic bacteriacontent, feaces are cultured on blood agar plates.

Results

The assays described above are used to determine the effectiveness ofcompositions comprising AP in vivo. Reductions in cytokine secretion areobserved; decreases in TNF alpha, Il-1 beta and IFN gamma levels aremeasured in the inflamed intestines upon oral administration of thealkaline phosphatase rich milkfat globule membrane fraction of bovinemilk.

Example 2 AP-Treated Mice Develop Less Severe Colitis after TNBS or DSSTreatment

Materials and Methods:

Experimental Design:

Three independent experiments were performed. For the first DSSexperiment 42 eight-week old wild type C57BL/6 mice were obtained andfor the TNBS experiment 20 eight-week old wild type BALB/c mice wereobtained, from Charles River and from Harlan Nederland (Horst, TheNetherlands), respectively. For a second DSS experiment, 72 eight weekold C57BL/6 mice were obtained from Charles River Nederland. During theexperiments, the mice were housed under standard conditions and theywere allowed free access to water and food.

In the first experiment with C57BL/6 mice, colitis was induced byadministration of 1.5% (n=18) or 2.5% (n=20) dextran sulphate sodium(DSS) in the drinking water of the mice for one week.

In the BALB/c mice, colitis was induced by rectal administration at dayzero and seven of 1 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS)(Sigma Chemical Co, St. Louis, Mo., USA) dissolved in 40% ethanol in PBSusing a vinyl catheter that was positioned three centimetres from theanus. Preceding to the instillation, the mice were anaesthetised usingisoflurane(1-chloro-2,2,2,-trifluoroethyl-isofluranedifluoromethyl-ether) (AbbottLaboratories Ltd., Queen-borough, Kent, UK) and after the instillationthe mice were kept vertically for 30 seconds. After 48 hours of thesecond TNBS administration, the mice were sacrificed.

During the induction of colitis, ten BALB/c and twenty C57BL/6 micereceived orally 100 Units of alkaline phosphatase solved in 100 μl of100 mM Tris (pH 7.8) once a day; the other mice received exclusively 100μl of 100 mM Tris (pH 7.8). Four C57BL/6 mice were used as a referencecontrol: they got no colitis and no treatment.

For a second DSS experiment, colitis was induced in C57BL/6 mice (n=48)by administration of 2% DSS in the drinking water for 5 days. 24 ofthese mice received 100 Units of alkaline phosphatase in 250 μl of 100mM Tris (pH 7.8) once a day from day 5 up to day 14, whereas the other24 mice received vehicle alone. A group of 24 mice that received normaldrinking water and vehicle only served as reference control. This setupwas used to investigate the use of AP as a rescue drug, once colitis isestablished.

In all experiments, the weight and temperature of the mice were recordeddaily. After sacrificing the mice, caudal lymph node (CLN) and colonwere obtained from the mice. Through a midline incision, the colons wereremoved and opened longitudinally. After removing the faecal material,the weight of the colons was measured and used as an indicator ofdisease-related intestinal thickening. The colons were divided in twoparts, one of which was used for histological analysis and the other forcytokine detection.

Histological Analysis:

The longitudinally divided colons were fixed in 4% formaldehyde embeddedin paraffin for routine histology. Three transverse slices (5 μm), takenfrom each colonic sample, were stained with haematoxylin-eosine andexamined by light microscopy. Colonic inflammation was evaluated in ablind manner by estimating the 1) percentage of involved area, 2) theamount of follicles, 3) oedema, 4) fibrosis, 5) erosion/ulceration, 6)crypt loss and 7) infiltration of granulocytes and 8) monocytes with amaximal score of 26.

The percentage of area involved and the crypt loss was scored on a scaleranging from 0 to 4 as follows: 0, normal; 1, less than 10%; 2, 10%; 3,10 to 50%; 4, more than 50%. Follicle aggregates were counted and scoredas follows: 0 point, 0-1 follicles; 1 point, 2-3 follicles; 2 point, 4-5follicles; 3 point, more than 6 follicles. Erosions were defined as 0 ifthe epithelium was intact, 1 for ulceration that involved the laminapropria, 2 ulcerations involving the submucosa, and 3 when ulcerationswere transmural. The severity of the other parameters was scored on ascale 0 to 3 as follows: 0, absent; 1, weak; 2, moderate; 3, severe.

Cell Culture:

Caudal lymph node cells of TNBS mice were isolated by passing the lymphnode through a 40 μm filter cell strainers (Becton/Dickson Labware, NewJersey, USA). The isolated lymphocytes were suspended in 4 ml RPMI 1640medium, including L-glutamine, 10% foetal calf's serum (FCS) andantibiotics (Penicillin G sodium 10000 U/ml, Streptomycin sulphate 25μg/ml. Amphotericin B 25 μg/nl; Gibco/BRL, Paisley, Scotland). The cellswere counted and added to flat-bottom 96-well plates at 2×105 cells perwell in a total volume of 200 μl of the same medium. The cells werecultured in the presence of immobilised α-CD3 (1:30 concentration;145.2C11 clone) and soluble α-CD28 (1:1000 concentration; PharMingen)for 48 hours at 37° C. The supernatant was collected and used for acytokine bead as-say (CBA).

Homogenisation and Enzymatic Determination

Swiss roles of colonic samples that were taken 6 cm from the anus werefrozen in the nitrogen. Homogenates were made with a tissue homogeniserin 9 volumes Greenberger lysis buffer (300 mmol/L NaCl, 15 mmol/L Tris,2 mmol/L MgCl2, 2 mmol/L Triton X-100 (Sigma, St. Louis, Mo.), PepstatinA, Leupeptin, Aprotinine (Roche, Mannheim, Germany), all 20 ng/mL; pH7.4). The tissue was lysed for one hour on ice and centrifuged for 7minutes at 3000 rpm and for 10 minutes at 14000 rpm. The supernatant wascollected and stored frozen until the day of enzymatic determination andBD cytokine bead array analysis.

The faeces of the mice were collected during the period of colitis. Theweight of the faeces was recorded and suspended in 0.6 ml of 50 mMglycine buffer with 0.5 mM MgCl2 (pH=9.6 at 25° C.). Aftercentrifugation (10′, 13000 rpm), the supernatant was stored frozen untilthe day of determination of alkaline phosphatase activity.

The activity of alkaline phosphatase in the colon and faeces wasmeasured spectro-photometrically, using p-nitrophenyl phosphate as asubstrate in 50 mM glycine buffer with 0.5 mM MgCl₂ (pH=9.6 at 25° C.).Enzymatic activity was expressed in mU/ml for the colon homogenates andin mU/mg for the faeces.

Cytokine Bead Assay (CBA)

A cytokine bead assay was performed to determine simultaneously theproduction of TNF-α, IFN-γ, IL-2, IL-4 and IL-5 in colon homogenates andCLN cell culture supernatant according to the manufacturersrecommendations of Becton Dickinson (BD). Briefly, particles(polystyrene beads) were dyed to five fluorescence intensities. Theproprietary dye had an emission wavelength of ˜650 nm (FL-3). Eachparticle was coupled via a covalent linkage based on thiol-maleimidechemistry with an antibody against one of the five cytokines andrepresented a discrete population, unique in their FL-3 intensity. TheAb-particles served as a capture for a given cytokine in theimmuno-assay panel and could be detected simultaneously in a mixture.The captured cytokines were detected via direct immunoassay using fivedifferent antibodies coupled to phycoerythrin (PE), which emitted at˜585 nm (FL-2). The standards ranging from 0 to 2000 pg/ml were mixturesof all five cytokines, so that five standard curves were obtained. Foreach sample and cytokine standard mixture, 10 μl of capture Ab-beadreagent, samples or standard and detector Ab-PE reagent were incubatedfor three hours and were washed to remove unbound detector Ab-PE reagentbefore data acquisition using flow cytometry. Two-colour flow cytometricanalysis was performed using a FACScan® flow cytometre (Becton DickinsonImmunocytometry Systems (BDIS), San Jose, Calif.). Data were acquiredand analysed using Becton Dickinson Cytometric Bead Array (CBA)software.

Statistical Analysis

All data were expressed as the means±the standard deviation. Whereindicated Student's t test was used to calculate statisticalsignificance for difference in a particular measurement betweendifferent groups. Values of p<0.05 were considered statisticallysignificant (*p<0.05).

Results

To investigate whether oral AP has therapeutic potential in IBD, theTNBS- and DSS-induced colitis in mice were used as models. As predicted,intrarectal instillation of TNBS and oral administration of DSS resultedin diarrhoea and wasting disease. At day two, two of the ten mice ineach of the two treatment groups died (see FIGS. 2 and 3) indicatingthat AP does not prevent mortality due to the early inflammatoryresponse Two or three days after the initial intrarectal administrationof TNBS a delayed hypersensitive responsive type 4 reaction is activatedand causes most of the inflammation, which leads to additionalmortality. Although AP treatment could not prevent the death of the miceduring this first reaction, it prevented additional deaths due to thesecondary inflammatory response.

In the groups that received 2% DSS in their drinking water for 5 dayswith subsequent AP gavage for up to 14 days, 33% of the mice diedbetween day 8 and 10 in the AP treated group. Thereafter no mice died inthis group. In the placebo treated group, however, in total 60% of themice died from day 8 onwards until day 14. Similar to the TNBS model, APis able to reduce mortality in the later stage of DSS-colitis, but notin the induction phase.

After the first administration of TNBS the weight of the mice hasdecreased at the first day in both the AP-treated as in the control mice(see FIG. 4). The weight of the control mice, however, was decreased to92% of their initial weight at day four, whereas the weight of theAP-treated mice was increased to above 98% of their initial weight andwas stabilised. At the sixth day, the control mice also reached theirinitial weight. The effects on weight are thus almost synchronized tothe described survival benefits.

In the DSS studies, only the second group that received 2% DSS and wasfollowed up to 14 days showed statistical significant differences ondays 10, 11 and 12 (FIG. 5). The other two groups were followed only upto day 7 and showed, as the former group, no difference between APtreated and untreated groups until that day (data not shown).

Example 3 Cytokine Production and AP Activity were Decreased in Colonsof AP-Treated Mice

Colon homogenates of TNBS and DSS mice were analysed for the productionof cytokines by a cytokine bead assay to investigate the size of the Th1response. In contrast to the increased cytokine production in the CLN ofAP-treated mice, the production of TNF-α, IFN-γ, IL-2, IL-4 and IL-5 wasdecreased in the colon homogenates of these mice compared to the controlmice, however not significantly (see table 1). The mice with 2.5%DSS-induced colitis confirm similar results, although these results didnot reach statistical significance, too. (Data not shown).

TABLE 1 Cytokine concentrations in colon homogenates measured by CBAControl TNBS mice AP-treated TNBS mice TNF-α 19.7 ± 12.4 10.8 ± 10.8IFN-γ 8.2 ± 5.5 4.0 ± 4.6 IL-2 4.4 ± 2.9 2.7 ± 2.6 IL-4 15.0 ± 7.0  8.9± 7.5 IL-5 5.9 ± 4.0 2.9 ± 3.8 Control 1.5% DSS mice AP-treated 1.5% DSSmice TNF-α 15.6 ± 5.1 13.1 ± 5.4  IFN-γ 100.5 ± 82.3 28.4 ± 19.8 Control2.5% DSS mice AP-treated 2.5% DSS mice TNF-α  89.3 ± 122.3 60.7 ± 40.8IFN-γ 130.0 ± 85.5  83.2 ± 90.0 IL-2 13.2 ± 25.8 7.2 ± 6.0 IL-4 23.4 ±42.5 24.7 ± 19.2 IL-5 19.5 ± 36.8 13.9 ± 12.5The production of IL-2, -4 and -5 in the 1.5% DSS mice were almostundetectable ^((data not shown)). The production of TNF-α was decreasedin the AP-treated mice compared to the DSS control mice, however notsignificantly (see FIG. 7). In case of the IFN-γ production, thedifferences were significant (p<0.05): the IFN-γ production wasdecreased from 100.5±82.3 pg/ml in DSS control mice to 31.6±21.3 pg/mlin AP-treated mice.

Example 4 Single Oral Dose Pharmacokinetic Assay of BIAP in Mice

Materials and Methods

Subject of investigation was the local and systemic bioavailabilityafter single high dose application of oral BIAP (Bovine intestinalalkaline phosphatase). The test material was delivered as a solution andwas stored at 4° C. until use. Dosing dilutions in autoclaved drinkingwater were prepared freshly on the day of treatment. Autoclaved drinkingwater was used as control solution.

The black/6 mouse is a suitable rodent species for DSS-induced colitisand is acceptable to regulatory authorities for safety testing. The oralroute of administration corresponds to the intended therapeutic use inhumans.

The study room and cages were cleaned and disinfected. During the study,the room and cages were cleaned at regular intervals. The roomtemperature was adjusted to 22±3° C. and the relative humidity was keptbetween 30% and 70%. These parameters were monitored daily. Artificiallight was set to give a cycle of 12 hours light and 12 hours dark withlight on at 6:00 a.m. Air was changed about 8 times per hour in theanimal room and filtered adequately. The animals were fed ad libitumwith SDS D3 pellets, analysed by the supplier for nutrients andcontaminants. Drinking water sterilized by autoclaving was continuouslyavailable ad libitum via drinking bottles. Consumption is controlledvisually on a daily basis.

TABLE 2 Experimental groups and DSS pretreatment were assigned accordingto the following table: Conc in No. of Card drinking Volume GroupTreatment animals labeling water [ml/kg b.w.] Red DSS 29 A-E (red) 2%See Table 3 green — 29 A-E (green) — See Table 3

TABLE 3 Experimental groups and BIAP treatment were assigned accordingto the following table: Conc in Volume Sacr. From Treat- No. of drinking[ml/kg Time Group Cage ment animals water b.w.] (min) Control A A greenPlacebo 5 — 12.5 150 Control B B green BIAP 3 6000 U/ml 12.5 10 ControlC B green BIAP 3 6000 U/ml 12.5 30 Control D C green BIAP 3 6000 U/ml12.5 60 Control E C green BIAP 3 6000 U/ml 12.5 90 Control F D greenBIAP 3 6000 U/ml 12.5 120 Control G D green BIAP 3 6000 U/ml 12.5 180Control H E green BIAP 3 6000 U/ml 12.5 240 Control I E green BIAP 36000 U/ml 12.5 360 DSS A A red Placebo 5 — 12.5 150 DSS B B red BIAP 36000 U/ml 12.5 10 DSS C B red BIAP 3 6000 U/ml 12.5 30 DSS D C red BIAP3 6000 U/ml 12.5 60 DSS E C red BIAP 3 6000 U/ml 12.5 90 DSS F D redBIAP 3 6000 U/ml 12.5 120 DSS G D red BIAP 3 6000 U/ml 12.5 180 DSS H Ered BIAP 3 6000 U/ml 12.5 240 DSS I E red BIAP 3 6000 U/ml 12.5 360

According to table 3 all mice except those in the placebo groups in bothcontrol and DSS treated groups received a single oral dose of 75,000U/kg BIAP per oral gavage (in 250 μl autoclaved drinking water). Placebogroups (n=5) received 250 μl autoclaved drinking water only. All animalswere treated once. Before treatment with BIAP, some of the animals weretreated with DSS according to table 2.

After blood collection, intestines from the animal were prepared andfaeces collected from each one third part of the small intestine andfrom the colon separately. Each sample was dissolved by vigorousvortexing in 1 ml glycine buffer (25 mM; pH=9.6)

These samples were analyzed for alkaline phosphatase content using thean assay for the determination of alkaline phosphatase activity.Paranitrophenyl phosphate, which is colourless, is hydrolysed byalkaline phosphatase at pH 9.6 and 25° C. to form free paranitrophenol,which is coloured yellow. The reaction can be followedspectrophotometrically. The change in optical density at 405 nm per unittime is a measure of the alkaline phosphatase activity. The amount ofenzyme causing the hydrolysis of one micromole of paranitrophenylphosphate per minute at pH 9.6 and 25° C. is defined as one unit. Theamount of units present in a sample can be calculated or calibratedagainst a curve of AP samples with a known AP concentration.

Results

During the pretreatment phase with DSS, mice were weighed daily as anindication for development of colitis. Weight loss of about 10% comparedwith control mice is used to identify DSS induced colitis. On day 6,mice that were treated for 5 days with DSS had lost around 10%bodyweight (FIG. 8) and were used in the pharmacokinetic experiment toidentify local and systemic bioavailability of administered BIAP duringcolitis. One day later, control mice were used in the same manner.

In order to analyze DSS uptake, drinking bottles were weighed dailybefore and after refilling of the bottles. The first three days, DSStreated animals consumed approx. the same amount of water as controlanimals did. Thereafter, DSS mice drank less, probably as a result ofdecreased well being. However, daily intake of DSS was about 40mg/mouse, which was shown to be sufficient for induction of colitis inother experiments (see FIG. 9).

The objective of this study was to analyze the local bioavailability inthe intestinal tract. For this purpose, on different time points micewere sacrificed and the intestinal tract from duodenum to colonisolated. The small intestine (inc. Duodenum) was divided in two threeequal parts. Faeces from each part and from the colon was extracted andalkaline phosphatase activity measured. In FIG. 10, the alkalinephosphatase activity in the different parts of the intestine atdifferent time points is shown. No alkaline phosphatase could bemeasured at any time point in the duodenum and proximal part of thejejunun. This was probably due to the fact that passage through thispart takes place within 10 minutes, the first sampling point. At 10minutes both DSS as well as control animals have peak alkalinephosphatase levels in the distal part of jejunum and proximal part ofileum. Between half an hour and one hour, peak values of AP occur in thedistal part of the ileum. In the colon, alkaline phosphatase isincreased one and a half hour after gavage and after six hours, mostalkaline phosphatase was either excreted or broken down.

Considering that a total of 1350 U was administered per mouse, localbioavailability is estimated in the distal part of the ileum and thecolon at 200/1350=15% and 80/1350=6%, respectively. However these valuesare probably underestimation as they are based on mean peak values of 3individual mice each (see discussion).

The objective of this study was to estimate the local bioavailability ofBIAP after high oral dose administration. The local bioavailability wasestimated at 15% in the distal part of the ileum and 6% in the colon.These values are probably grossly underestimated, because values arefrom individual mice at a given time point and do not represent alkalinephosphatase activity throughout the tract of one mouse followed in time.As a consequence, peak values may be missed in individual mice in agiven part of the intestine at a given time point. However, the resultsas depicted in FIG. 10 clearly demonstrate local bioavailability of APupon oral administration. These results underscore the feasibility ofthe method of the current invention to administer to a subject a sourceof alkaline phosphatase in order to prevent or reduce (toxic) LPS influxthrough a mucosal lining of a mammalian body cavity.

FIGURES

FIG. 1, explains the model of the current invention in three stages, 1.Mucosa in healthy condition, with normal AP histochemistry and LPSdetoxification. 2. Diseased condition, deficient AP staining,insufficient detoxification of LPS by AP, influx/translocation of toxicLPS from the gut into circulation leading to an inflammatory response 3.Restoration of mucosal AP levels and detoxification of LPS by providingan exogenous source of AP.

FIG. 2, case fatality rate in mice with TNBS-induced colitis

FIG. 3, case fatality rate in mice with DSS-induced colitis

FIG. 4, Weight loss in AP-treated and non-treated mice with TNBS-inducedcolitis.

FIG. 5, Weight loss in AP treated and non treated animal withDSS-induced colitis

FIG. 6, Cytokine production in the colon of mice with TNBS-colitis.

a. The TH1 response in the colon

b. The TH2 response in the colon

FIG. 7, The concentration of a. TNF-α and b. IFN-γ in colon homogenates.The control mice are normal values of non-ill and non-treated mice.

FIG. 8, DSS treatment decreases bodyweight. Mice were treated eitherwith normal drinking water or with drinking water containing 2% DSS for5 days. On day six, DSS treated animals had lost around 10% bodyweightcompared with control animals and were defined ‘colitic’. Shown are meanwith standard deviations of 29 animals per group.

FIG. 9, DSS treated mice consume between 40-80 mg DSS per day. Mice weretreated either with drinking water or drinking water containing 2% DSS.Drinking bottles were weighed before and after refilling. Values showmean water intake in g/mouse/day.

FIG. 10, Alkaline phosphatase levels in different parts of intestinaltract at different time points after gavage. At different time pointsafter gavage, mice were sacrificed and intestines removed. Faeces fromdifferent parts of the tract were collected and alkaline phosphatasecontent measured. Shown are mean values of three mice per time point.

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The invention claimed is:
 1. A method for preventing or reducing LPStoxicity at an intestinal mucosa in a subject, comprising administeringa composition comprising a source of alkaline phosphatase (AP) that issuitable for preventing or reducing lipopolysaccharide (LPS)-inducedtoxicity at the intestinal mucosa to a subject suffering from agastro-intestinal (GI) tract inflammatory disease selected from thegroup consisting of Crohn's disease, enterocolitis, and ulcerativecolitis, wherein the AP is delivered to the GI tract of the subject totreat said GI tract inflammatory disease.
 2. The method according toclaim 1, wherein the subject suffers from an LPS-mediated orLPS-exacerbated disease or condition.
 3. The method according to claim 1wherein the composition is administered orally.
 4. The method accordingto claim 1, wherein the GI tract inflammatory disease is Crohn'sdisease, or ulcerative colitis.
 5. The method according to claim 1wherein the GI tract is more sensitive to LPS as a result of enhancedmucosal permeability of LPS due to (i) decreased intestinal perfusion or(ii) intestinal ischemia.
 6. The method according to claim 5 wherein thedecreased perfusion or ischemia is a result of cardiopulmonary bypasssurgery, trauma or wounding, burns, cardiac surgery, congenital heartdisease, congestive heart failure, coronary heart disease, or ischemicheart disease.
 7. The method according to claim 1 wherein thecomposition is administered topically to said mucosa.
 8. A methodaccording to claim 1 wherein the composition further comprises apharmaceutically acceptable: (i) stabilizer, (ii) activator, (iii)carrier, (iv) permeator, (v) propellant, (vi) disinfectant, (vii)protectant, (viii) diluent, (ix) nutrient or (x) another excipient, thatpromotes AP delivery to said mucosa.